Imaging of tissues

Light sheet imaging was carried out on a home-made system (Supplementary Figure 2). A diode-pumped solid-state laser (LMM-GB1, Laserglow Technologies, Toronto, Canada) was used as the 532 nm illumination source. The initial diameter of laser beam was 2 mm, and then light passed through a 5x achromatic beam expander (GBE05-A, Thorlabs Inc, New Jersey, USA). A beam splitter (BS013, Thorlabs Inc, New Jersey, USA) was employed to form dual-illumination onto the sample from opposite directions. Each beam was focused by a plano-convex cylindrical lens (f = 50 mm, LJ1695RM-A, Thorlabs Inc, New Jersey, USA) and was then reshaped by a group of achromatic doublets (AC254-060-A, AC254-100-A, Thorlabs, New Jersey, USA). After passing through an f = 150 mm lens, the beam expanded to a sheet with the width of 40 mm, as well as the waist of 17 μm. The detection module was installed perpendicular to the illumination plane, and it was composed of a stereo microscope (MVX10, Olympus, Japan) with a 1x magnification objective (Numerical aperture, NA: 0.25), a scientific CMOS (ORCA-Flash4.0LT, Hamamatsu, Japan) and optical filters (Semrock, New York, USA). The sample with RIMS and 1% agarose solution was embedded in a borosilicate glass tubing with an inner diameter of less than 6 mm, and then the mounted sample was placed on a motorized translational stage. Both the sample and its holder, but not the translational stage, were immersed in a transparent chamber filled with 99.5% glycerol to match the refractive index among different materials. Illumination and detection were controlled by a computer with dedicated SSD RAID 0 storage for fast data streaming.

Colorimetrically labeled tissues were imaged using a Zeiss Axiovert inverted microscope with the included epi-illumination light source. Brain sections labeled with Alexa 594-labeled secondary antibodies were imaged using this Zeiss Axiovert microscope with a xenon-mercury arc lamp and appropriate excitation and emission filters. Images were collected using a Photometrics CoolSnap HQ2 camera.